The role of the endothelium in immune reactions is greatly amplified when endothelial cells (EC) express MHC class II antigens and are able to present antigens to CD4+ T cells. Increased expression of MHC class II antigens on vascular endothelium is a common observation in allografts undergoing acute rejection and is generally ascribed to the host immune response directed against graft alloantigens. This is viewed not only as a sign of rejection, but also as a major amplifying stimulus of the rejection process. The comprehensive objective of this research is to study the molecular mechanisms of intercellular contact-dependent endothelial class II HLA induction by lymphocytes, in particular natural killer (NK) cells. We have previously demonstrated that CD56+, CD3- NK cells are the most efficient cellular inducers of EC HLA-DR and that intercellular adhesion is a critical component of this phenomenon. Gamma interferon (IFNgamma) does not appear to play a major role in this phenomenon despite the fact that it is the only recognized inducer of EC class II MHC antigens, and tyrosine phosphorylation is involved. Our hypothesis is that there exists an alternative, IFNgamma-independent pathway which, as a consequence of NK (and perhaps CD8+) lymphocyte-EC contact, efficiently induces endothelial class II HLA gene transcription. Specific proposals now include to (1) compare patterns of IFNgamma- and NK cell contact-induced tyrosine phosphorylated EC proteins, and evaluate whether tyrosine kinase inhibition modulates induced DNA binding activity to critical DRalpha promoter regions; (2) investigate the role of lymphocyte receptors, EC ligands and allorecognition in the contact- dependent activation events, utilizing newly generated monoclonal antibodies, anti-EC NK clones, and alpha-L beta-2 (LFA-1) transfectants to address the transmembrane signalling potential of EC ICAM-1; (3) dissect the molecular genetic controls of NK cell- and IFNgamma-mediated HLA- DRalpha gene activation utilizing (a) transfected mutant DRalpha promoter constructs to examine CIS regulatory elements, (b)electrophoretic ability shift assays with target sequences derived from the promoter mapping, (c) antisense oligonucleotides based on translational disruption directed at candidate inducible transacting factors in EC, and (d) EC cDNA library screening for relevant DRalpha transcription/DNA binding factors, also with sequences derived from the mutant promoter analyses; and (4) assess the role of NK cells in allograft endothelial activation and injury in vivo, utilizing a SCID mouse model engrafted with human skin and lymphocytes, enriched or, depleted,of NK cells. This work may provide direction for pharmacologic and/or genetic manipulations, in vivo, to interfere with endothelial activation and immune-mediated vasculopathy, including allograft rejection as well as certain forms of vasculitis and arteriosclerosis.